Polymorphonuclear leukocyte dysfunction syndrome in patients with increasing sepsis severity

Production of oxygen radicals is required for both microbicidal and tissue-toxic effector functions of granulocytes. Inasmuch as an ambivalent role of polymorphonuclear leukocytes (PMNs) may become apparent during sepsis, we studied levels of hydrogen peroxide (H2O2) production by PMNs depending upon the nature of different particulate and soluble stimuli in patients with increasing sepsis severity. Patients with sepsis (n = 15), severe sepsis (n = 12), or septic shock (n = 33) were prospectively enrolled in the study. Healthy volunteers of comparable age and sex served as controls (n = 50). Unopsonized and opsonized zymosan particles were used to assess adhesion, phagocytosis, and the associated H2O2 production. Zymosan particles are rich in beta-glucans and lectin structures that are known to trigger H2O2 production via two major non-toll-like receptor pathogen recognition receptors, comprising the lectin-binding site in the alpha-chain (CD11b) of the complement receptor type 3 and the more recently identified nonclassical C-type lectin, dectin-1. To determine H2O2 production upon cell activation by soluble stimuli, PMNs were activated by the chemotactic tripeptide (N-formyl-methionyl-leucyl-phenylalanine [fMLP]) alone or after priming of cells by preincubation with tumor necrosis factor alpha. To get insight into the changes of fMLP receptor classical intracellular signaling pathways, PMNs were also incubated with the calcium ionophore A23187 and the phorbol ester phorbol myristate acetate, bypassing receptor-dependent signal transduction to directly activate calcium/calmodulin kinase- and protein kinase C-dependent pathways, respectively. As compared with healthy volunteers, levels of H2O2 production by PMNs from septic patients varied depending upon the nature of the activating signal: reduced (zymosan), unchanged (phorbol myristate acetate, opsonized zymosan), and enhanced (spontaneous, fMLP, fMLP + tumor necrosis factor alpha, A23187), with the changes most pronounced in patients with septic shock. Specifically, phagocytosis of zymosan and the associated H2O2 production were significantly decreased whereas spontaneous and stimulated H2O2 production elicited by soluble stimuli strongly increased. Thus, these findings suggest the development of a PMN dysfunction syndrome in patients with increasing sepsis severity. Moreover, as binding of zymosan particles to the PMNs' surface remained unchanged despite increasingly suppressed phagocytosis and associated H2O2 production, observed effects are likely to reflect defects in signaling by the lectin-binding site of CD11b and/or the beta-glucan receptor dectin-1, respectively.     

Cytochalasin B facilitates the inhibition of human polymorphonuclear leukocyte generation of superoxide by verapamil

Several functions of the human neutrophil are dependent upon extracellular calcium for optimal activation. We studied the calcium dependency of human polymorphonuclear leukocyte (PMN) superoxide generation in response to opsonized zymosan particles and its modulation by the calcium channel antagonist verapamil. PMNs were isolated from anticoagulated blood after Ficoll-Hypaque density centrifugation. The isolated PMNs were incubated with opsonized zymosan particles. The superoxide anion generated was measured by a cytochrome C reduction assay. When PMNs were incubated with opsonized zymosan in a calcium-free buffer, there was 0.45 +/- 0.06 nmol of cytochrome C reduced per 1 X 10(6) PMN per minute. This increased to 0.76 +/- 0.12 nmol per 1 X 10(6) PMN per minute when 0.6 mmol/L Ca++ was present. Moreover, if the PMNs were incubated with cytochalasin B (5 micrograms/ml), the generation of superoxide anion was further enhanced. If the same experiments were conducted in the presence of verapamil, 100 mumol/L, superoxide generation was inhibited by 51.5% +/- 8.4%. For verapamil to inhibit superoxide generation, cytochalasin B was necessary. Our results suggest that verapamil-sensitive calcium channels may exist in human PMNs, and the demonstration of their presence is cytochalasin B-dependent.     

Purine catabolism in polymorphonuclear neutrophils. Phorbol myristate acetate-induced accumulation of adenosine owing to inactivation of extracellularly released adenosine deaminase.

Since physiological concentrations (0.1-1 microM) of adenosine influence the functions of human polymorphonuclear neutrophils (PMNs), we investigated the metabolism of adenosine in suspensions of stimulated and unstimulated PMNs. Stimulation with phorbol myristate acetate (PMA, 1 microM), but not by zymosan (0.5 mg/ml) or N-formyl-methionyl-leucyl-phenylalanine (fMLP, 1 microM), provoked an accumulation of endogenous adenosine at a rate of 2.3 +/- 1.0 amol/cell per minute. A similar accumulation was observed with both unstimulated and stimulated PMNs after the addition of deoxycoformycin (dCF, 1-100 microM), an inhibitor of adenosine deaminase. Exogenous adenosine (10 microM) was deaminated at a rate of 9.8 +/- 3.7 amol/cell per minute in control or zymosan or fMLP-stimulated PMN suspensions. This deamination was nearly completely suppressed when the PMNs had been stimulated with PMA. In contrast, the activity of adenosine deaminase in PMN lysates (231 +/- 72 amol/cell per minute) was not modified by PMA stimulation. alpha, beta-Methyleneadenosine 5'-diphosphate (AMPCP, 2.5 mM), an inhibitor of membranous ecto-5'-nucleotidase, profoundly inhibited endogenous adenosine accumulation under all conditions. PMA stimulation also provoked an inactivation of extracellular adenosine deaminase, purine nucleoside phosphorylase, and lactate dehydrogenase in PMN suspensions. We concluded that PMNs, even when not stimulated, continuously produce adenosine by dephosphorylation of extracellularly released adenylates; and that stimulation of PMNs by PMA causes adenosine accumulation owing to the inactivation of adenosine deaminase released by broken cells.     

Electrochemical detection of nitric oxide production in human polymorphonuclear neutrophil leukocytes.

The detection of nitric oxide (NO) release by human polymorphonuclear neutrophil leukocytes (PMNs) presents several difficulties, mainly due to concomitant production of O2- and H2O2, which could interfere with the measurements. A Nafion and nickel porphyrin-coated microelectrode was used to measure NO production in PMNs in vitro. It allowed detection of 6.3 +/- 1.9 nM NO in a PMN-containing system and was unaffected by added chemicals. Addition of the chemotactic oligopeptide f-met-leu-phe (fMLP; 100 nM) induced a NO release which reached a value of 71 +/- 30 pmol NO/10(6) PMN x ml(-1) 5 min after stimulation in the presence of SOD (150 U/ml). If SOD was omitted, the corresponding value was 36 +/- 20 pmol NO/10(6) PMN x ml(-1). Presence or absence of catalase did not alter the amount of NO measured. Addition of the NO-synthase inhibitor N(G)-monomethyl-L-arginine (LNMMA; 1 mM) reduced the current by 82 +/- 20%. These results agree with the rate of NO production in human PMNs when measured spectrophotometrically using the NO-dependent oxidation of oxyhaemoglobin to methaemoglobin. The NO production in human PMN was dependent on fMLP concentrations, but independent of cell-concentrations of 0.5-3.5 x 10(6)/ml. This paper shows that a electrochemical method, e.g. Nafion and porphyrin-coated microelectrode, is suitable for studies of NO release from stimulated human PMNs.     

Increased neutrophil adenosine a3 receptor expression is associated with hemorrhagic shock and injury severity in trauma patients

Hypertonic saline (HS) has been investigated as an immune modulator following hemorrhagic shock and sepsis. The polymorphonuclear neutrophil (PMN) response to HS is regulated by the release of ATP, which is converted to adenosine and activates adenosine receptors. Binding to A3 adenosine receptors promotes PMN activation, and inhibition of A3 receptors improves the efficacy of HS resuscitation. A3 receptor expression of PMNs has not been previously evaluated in injured patients. Whole blood was obtained from 10 healthy volunteers and 60 injured patients within 2 h of injury. Inclusion criteria were blunt or penetrating injury with evidence of hypovolemic shock (systolic blood pressure [SBP] ≤90 mmHg and base deficit ≥6 mEq/L or need for blood transfusion) or evidence of severe traumatic brain injury including initial Glasgow Coma Scale score of 8 or less or evidence of traumatic brain injury on head computed tomography scan (head Abbreviated Injury Score ≥3) or intubation in the field or emergency department. A3 receptor expression was assessed by flow cytometry. Polymorphonuclear neutrophils were also exposed to fMLP or HS (20-40 mM) in vitro. Clinical data were collected including admission physiology, injury severity (Injury Severity Score [ISS]), development of multiple organ failure, and survival. In normal volunteers, less than 1% of PMNs expressed A3 receptors on the cell surface. A3 receptor expression was significantly higher in injured patients, and the level of expression correlated with the severity of injury (ISS ≥25: A3 positive PMN 36.6% vs. ISS <25: 16.2%; P = 0.019) and degree of hypovolemic shock (SBP ≤90 mmHg: A3 positive PMN 43.8% vs. SBP>90 mmHg: 20.6%; P = 0.008). Stimulation with fMLP or HS increased A3 expression in normal volunteers, but only in patients with ISS of less than 25 or without hypovolemic shock. A3 receptor expression on the surface of PMNs is upregulated by injury, and increased expression levels are associated with greater injury severity and hypovolemic shock. Hypertonic saline increases A3 expression of PMNs from healthy volunteers and less severely injured patients.     

Platelets as inhibitory cells in neutrophil-mediated cytolysis

Neutrophilic polymorphonuclear leukocytes (PMNs), triggered by opsonized zymosan (OPZ), lysed chicken red blood cells as measured by a chromium 51 release method. The lysis was prevented by scavengers of hypochlorous acid. When platelets were added to the cytolytic system, a dose-dependent inhibition of the lysis was observed. Moreover, platelets lowered the HOCl recovery from OPZ-triggered PMNs. A positive linear relationship was found between the extent of the lysis mediated by and the amount of HOCl recovered from PMNs. Finally, both the inhibition of the lysis and the reduction of the HOCl recovery induced by platelets were prevented by pulsing platelets with carmustine (BCNU) to block their glutathione cycle. These results suggest that platelets act by consuming via their glutathione cycle significant amounts of PMN-derived hydrogen peroxide, with a consequent impairment of the PMN HOCl production and, in turn, lytic efficiency. Consistent with such a conclusion, platelets were found to consume PMN-derived H2O2 via a BCNU-inhibitable process. Further, the platelet inhibitory activity could be abolished by the addition of an appropriate extra-flux of enzymatically generated H2O2. No evidence for a platelet-induced inhibition of OPZ-PMN interaction, PMN myeloperoxidase release, and H2O2 production was obtained. The present study provides direct evidence for a platelet-dependent mechanism capable of controlling the PMN production of highly reactive oxidants and, in turn, the PMN cytolytic activity.     

Glycogen metabolism in stored granulocytes

Optimal function of transfused granulocytes (PMNs) requires adequate glycogen metabolism. Previous studies in our laboratory suggested that stored PMNs had decreased glycogen. We report here the glycogen content and chemotaxis of stored PMNs, and the ability of fresh and stored PMNs to use glycogen as the fuel source for chemotaxis. PMNs were prepared from 8 fresh units of blood drawn into citrate-phosphate-dextrose-adenine, suspended at 2 or 8 X 10(7) PMN per ml in autologous plasma with or without 15 mM sodium bicarbonate, and stored at 22 to 24 degrees C in transfer packs for 48 hours. Glycogen was measured on resting PMNs, and after challenge with opsonized zymosan and F-Met-Leu-Phe (FMLP). The chemotaxis of fresh and stored PMNs was measured in the presence or absence of extracellular glucose. Fresh PMNs contained 10.3 +/- 0.5 (mean +/- SEM) micrograms of glycogen per 10(6) PMN. Glycogen decreased by 4.2 +/- 0.9 micrograms per 10(6) PMN after challenge with opsonized zymosan and by 1.1 +/- 0.6 micrograms per 10(6) PMN after FMLP. After 48 hours of storage, neutrophil glycogen increased by 18 percent, except in units stored at a concentration of PMN of 8 X 10(7) per ml without sodium bicarbonate. In PMNs from these units stored without bicarbonate, glycogen decreased by 9 percent (p less than .05), and there was a 19 and 55 percent decrease in the ability of PMN from these units to metabolize glycogen after exposure to opsonized zymosan and FMLP, respectively (p less than 0.05). In addition, in PMNs from units stored at a concentration of PMN of 8 X 10(7) per ml without bicarbonate, there was a 47 and 70 percent decrease in chemotaxis at 24 and 48 hours, respectively (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation of human neutrophil activation events by prolonged treatment with amiloride

Human polymorphonuclear leukocytes (PMNs) can be stimulated to release granule contents and to produce superoxide anion. These functional responses are associated with cellular alkalinization and influx of Na+ in exchange for H+. Amiloride is a potassium-sparing diuretic that will inhibit stimulus-induced Na+-H+ exchange and prevent an increase in cell pH. Amiloride has been shown to inhibit a number of protein kinases including the calcium phospholipid-dependent protein kinase. Because PMA, which binds and activates C-kinase, is a potent stimulus of the PMN, this study was undertaken to investigate the effect of prolonged incubation of PMNs with amiloride on PMN stimulation by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), the calcium ionophore A23187, opsonized zymosan particles, and the tumor promoter phorbol myristate acetate (PMA). Our results demonstrate that amiloride inhibits superoxide anion production by FMLP, A23187, and opsonized zymosan by causing a slower rate of release and lower maximal release without altering lag time. In contrast, amiloride, despite an inhibition of 22Na+ influx, did not affect superoxide anion production stimulated by PMA. PMN degranulation, phagocytosis, arachidonic acid release, and early influx of calcium were unaffected by preincubation with amiloride. These data suggest that PMN superoxide release induced by FMLP, A23187, and opsonized zymosan is likely modulated by amiloride-sensitive Na+-H+ exchange; and phorbol ester-induced superoxide anion release and degranulation by any stimulant do not appear to be modulated by inhibition of an amiloride-sensitive Na+-H+ exchange.     

Hypertonic saline inhibits neutrophil (PMN) priming via attenuation of p38 MAPK signaling

Priming of the neutrophil cytotoxic response is central to the pathogenesis of early postinjury multiple organ failure (MOF). Platelet-activating factor (PAF) has been implicated as a key inflammatory mediator in postinjury neutrophil priming and requires p38 MAPK signaling to produce its biologic effects. Hypertonic saline (HTS) resuscitation decreases the postinjury inflammatory response following shock in animals and decreases receptor-mediated neutrophil (PMN) cytotoxic functions in vitro. We hypothesized that HTS attenuates PAF priming of the PMN cytotoxic response by interfering with PAF-mediated p38 MAPK signal transduction. Isolated PMNs were preincubated in isotonic buffer or HTS (Na+ = 180 mM), then primed with PAF. Neutrophil CD11b/CD18 expression was measured by flow cytometry. Receptor-dependent (fMLP), N-formyl-methionyl-leucyl-phenylalanine, fMLP) and receptor-independent (PMA) O2- production was measured by reduction of cytochrome c in resting and PAF primed PMNs. Total p38 MAPK protein PAF-mediated p38 MAPK activation was assessed by western blot of PMN lysates. Clinically relevant levels of HTS attenuated PAF-mediated beta2-integrin expression. While HTS attenuated receptor-dependent (fMLP and PAF/fMLP) O2- production, receptor-independent (PMA) O2- production was unaffected. Conversely, HTS attenuated PAF priming of PMA-mediated O2- production. PAF and HTS did not alter total cellular p38 MAPK content. Clinically relevant levels of HTS alone did not activate p38 MAPK but inhibited PAF mediated p38 MAPK activation. HTS attenuates PAF priming of the PMN cytotoxic response by altering intracellular signal transduction. Therefore, HTS resuscitation may attenuate postinjury PMN priming and ultimately the risk of developing MOF.     

Neutrophil heterogeneity in patients with blunt trauma

To determine whether neutrophil (PMN) dysfunction observed in patients with blunt trauma could be explained by alterations in PMN functional subpopulations and to further study the origins of PMN heterogeneity, we studied PMN subpopulations in 18 patients with severe blunt trauma by using a micropore filter chemotactic assay and a mouse monoclonal antibody (31D8 Mab). A major PMN subpopulation binds 31D8 Mab avidly (31D8 "bright") and depolarizes and responds chemotactically to formyl peptide (fMLP) and C5a; a minor PMN subpopulation binds 31D8 Mab weakly (31D8 "dull") and fails to depolarize and responds poorly to fMLP and C5a. Fourteen patients with trauma had marked alteration of PMN 31D8 expression compared with healthy controls 52% +/- 20% versus 92% +/- 4% bright PMNs, respectively (p less than 0.01). These patients also had significantly decreased PMN chemotaxis and increased band counts compared with controls 30 +/- 10 micron versus 53 +/- 19 micron (p less than 0.01) and 34% +/- 14% versus 6% +/- 1% (p less than 0.01), respectively. Four patients with less-severe injuries had unaltered 31D8 PMN expression and normal PMN chemotaxis. In patients whose band counts exceeded 20%, there was a strong correlation between the number of bands and the percentage of 31D8 dull PMNs. PMNs that weakly express the 31D8 antigen appear to be less mature than PMNs that strongly express the antigen regardless of cell morphology (i.e., bands, multilobed cells). The data suggest that the decreased PMN chemotaxis and increased infection rate in patients with blunt trauma is caused partly by an increase in the number of poorly functioning 31D8 dull PMNs.     

Acute hypoxemia in humans enhances the neutrophil inflammatory response

The neutrophil (PMN) is regarded as a key component in the hyperinflammatory response known as the systemic inflammatory response syndrome. Acute respiratory distress syndrome (ARDS) and subsequent multiple organ failure (MOF) are related to the severity of this hyperinflammation. ICU patients who are at highest risk of developing MOF may have acute hypoxic events that complicate their hospital course. This study was undertaken to evaluate the effects of acute hypoxia and subsequent hypoxemia on circulating PMNs in human volunteers. Healthy subjects were exposed to a changing O2/N2 mixture until their O2 saturation (SaO2) reached a level of 68% saturation. These subjects were then exposed to room air and then returned to their baseline SaO2. PMNs were isolated from pre- and post-hypoxemic arterial blood samples and were then either stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or PMA alone, or they were primed with L-alpha-phosphatidylcholine, beta-acetyl-gamma-O-alkyl (PAF) followed by fMLP activation. Reactive oxygen species generation as measured by superoxide anion production was enhanced in primed PMNs after hypoxemia. Protease degranulation as measured by elastase release was enhanced in both quiescent PMNs and primed PMNs after fMLP activation following the hypoxemic event. Adhesion molecule upregulation as measured by CD11b/CD18, however, was not significantly changed after hypoxemia. Apoptosis of quiescent PMNs was delayed after the hypoxemic event. TNFalpha, IL-1, IL-6, and IL-8 cytokine levels were unchanged following hypoxemia. These results indicate that relevant acute hypoxemic events observed in the clinical setting enhance several PMN cytotoxic functions and suggest that a transient hypoxemic insult may promote hyperinflammation.     

Viricidal effect of polymorphonuclear leukocytes on human immunodeficiency virus-1. Role of the myeloperoxidase system.

Myeloperoxidase (MPO), H2O2, and chloride form an antimicrobial system in neutrophilic polymorphonuclear leukocytes (PMN) effective against a variety of microorganisms. Normal human PMN, when stimulated with phorbol myristate acetate or opsonized zymosan, are viricidal to HIV-1. The viricidal effect was lost when chloride was replaced by sulfate and was inhibited by the peroxidase inhibitor azide and by catalase, but not by heated catalase or superoxide dismutase, implicating H2O2. Stimulated PMN from patients with chronic granulomatous disease (CGD) were not viricidal to HIV unless H2O2 or glucose oxidase (which generates H2O2) was added, and the viricidal activity of H2O2-supplemented CGD PMN was inhibited by azide, implicating endogenous MPO. Stimulated PMN from patients with hereditary MPO deficiency had decreased viricidal activity unless MPO was added, and the viricidal activity of MPO-supplemented, MPO-deficient PMN was inhibited by catalase, implicating endogenous H2O2. The data suggest that when PMN are stimulated, MPO released by degranulation reacts with H2O2 formed by the respiratory burst to oxidize chloride to a product (presumably hypochlorous acid) that is toxic to HIV-1. Our findings raise the possibility that this viricidal effect of stimulated PMN may influence the host defense against HIV-1.     

Entry of lomefloxacin and temafloxacin into human neutrophils, peritoneal macrophages, and tissue culture cells.

The uptake of lomefloxacin (difluoroquinolone) and temafloxacin (trifluoroquinolone) by human polymorphonuclear leukocytes (PMNs), peritoneal macrophages (PM phis), and two tissue culture cells (McCoy and Vero) was measured by a fluorometric assay. Both antimicrobials reached high intracellular concentrations in PMNs [cellular to extracellular ratio (C/E) greater than 4], in PM phis (C/E greater than 3) and lower in tissue culture cells (C/E greater than 1) at an extracellular concentration of 5 mg/L. Lomefloxacin uptake by PMNs was more rapid than that of temafloxacin. Entry of both quinolones into PMNs was environmental temperature-dependent, but not affected by cell viability. Ingestion of opsonized Staphylococcus aureus did not affect the ability of PMNs to concentrate these antimicrobials. Ingestion of opsonized zymosan or stimulation with phorbol myristate acetate significantly increased the PMN association of both quinolones, this effect being particularly marked with temafloxacin. It is concluded that both lomefloxacin and temafloxacin are markedly concentrated within human phagocytes and tissue culture cells, although this phenomenon is not dependent on the degree of fluorination of the molecule.     

Blood transfusion and the two-insult model of post-injury multiple organ failure

Neutrophils (PMNs) have been implicated in the pathogenesis of multiple organ failure (MOF). The two-insult model of MOF is based on the fundamental concept that two sequential and independent insults that are individually innocuous against the host can cause overwhelming inflammation. The in vitro PMN priming/activation sequence simulates the two-insult model. Our work has demonstrated that transfusion is an early consistent risk factor for post-injury MOF and lysophosphatidylcholines (lyso-PCs) are generated in stored blood components. Additionally, platelet-activating factor (PAF) is a key inflammatory agent produced in severely injured patients. We therefore hypothesize that two events, trauma and transfusion, enhance PMN cytotoxicity irrespective of the sequence. Superoxide (O2-) production was measured by reduction of cytochrome c, adherence to fibrinogen was assessed by the radioactivity of adherent Na2(51)CrO4 (51Cr)-labeled PMNs, and endothelial cell (EC) damage by measuring the radioactivity released from 51Cr-labeled human umbilical vein endothelial cells monolayers. Isolated PMNs were primed with buffer, PAF (2 microM), or lyso-PCs (4.5, 15, and 30 microM) followed by activation with buffer, N-formyl-methionyl-leucyl-phenylalanine (fMLP) (1 microM), PAF (2 microM), or lyso-PCs (4.5, 15, and 30 microM). Neither PAF nor lyso-PCs alone stimulated O2- production. While PAF alone caused PMN adherence, lyso-PCs alone did not allowed PMNs to adhere to fibrinogen. However, both combinations of PAF/lyso-PCs and lyso-PCs/PAF significantly augmented O2- production and PMN adherence. Furthermore, these enhanced PMN cytotoxic responses significantly caused EC damage. These findings suggest that in the scenario of the two-insult model, early or late transfusion administered following trauma can provoke PMN cytotoxicity via priming or activation, thereby increasing the risk of post-injury MOF.     

Superoxide production by peripheral blood monocytes during sustained endotoxaemia in sheep.

1. We have studied the time course of the numbers of arterial monocytes and their superoxide anion (O2-) production in a chronically instrumented sheep model of subacute endotoxaemia induced by a continuous intravenous infusion of Escherichia coli lipopolysaccharide (20 ng min-1 kg-1). 2. Four out of 11 animals died from irreversible respiratory and cardiovascular failure within 21 h of the start of lipopolysaccharide administration ('non-survivors'), whereas in the seven surviving sheep ('survivors') there was a persistence of decreased systemic vascular resistance, systemic hypotension, pulmonary hypertension, anorexia and lethargy. 3. O2- generation by isolated monocytes was measured by the O2- dismutase-inhibitable reduction of ferricytochrome c after stimulation with phorbol myristate acetate (100 ng/ml) or opsonized zymosan (3 mg/ml). Basal mean value of phorbol myristate acetate-stimulated O2- production was significantly (P = 0.008) higher for non-survivors (31.3 +/- 8.8 nmol 30 min-1 10(-6) cells; n = 4) than for survivors (6.2 +/- 2.3 nmol 30 min-1 10(-6) cells; n = 7). 4. For both survivors and non-survivors, monocyte counts and phorbol myristate acetate-stimulated O2- production increased over time to reach in survivors a plateau after 2 days of continuous lipopolysaccharide infusion. Similar results were obtained when monocytes were stimulated for O2- production with opsonized zymosan. 5. These results suggest that (1) increased O2- production by monocytes and monocytosis appear with a precise, delayed time course during the development of subacute endotoxaemia in sheep; and (2) a high stimulated O2- production by monocytes before lipopolysaccharide administration may represent a predictive factor for the subsequent respiratory failure and outcome of endotoxaemia.     

Lipid complexing decreases amphotericin B inflammatory activation of human neutrophils compared with that of a desoxycholate-suspended preparation of amphotericin B (Fungizone).

Amphotericin B (AmB) has toxic effects and alters neutrophil (polymorphonuclear leukocyte [PMN]) function. A lipid-complexed formulation of AmB (AmB-LC) has been reported (A. S. Janoff, L. T. Boni, M. C. Popescu, S. R. Minchey, P. R. Cullis, T. D. Madden, T. Taraschi, S. M. Gruner, E. Shyamsunder, M. W. Tate, R. Mendelsohn, and D. Bonner, Proc. Natl. Acad. Sci. USA 85:6122-6126, 1988) to be less toxic than a desoxycholate-suspended preparation of AmB (AmB-des; Fungizone). In this study we compared the effects of AmB-des and AmB-LC on in vitro PMN function. Neither form of AmB stimulated PMN chemiluminescence, but AmB-des (2 micrograms/ml) nearly tripled PMN chemiluminescence in response to f-Met-Leu-Phe (fMLP), a phenomenon known as priming. Because AmB stimulates monocytes to release cytokines which can affect PMN function, we studied the effects of AmB on PMNs in mixed leukocyte cultures. AmB-des (1 to 2 micrograms/ml) increased the chemiluminescence of PMNs plus mixed mononuclear leukocytes (MNLs) to fMLP. The activity was about three times that of PMNs plus MNLs and seven times the activity of PMNs stimulated with fMLP in the absence of MNLs. Cell-free AmB-des (2 micrograms/ml)-stimulated, MNL-conditioned medium primed pure PMNs to a level equal to that of whole MNLs treated with AmB-des. AmB-LC was much less potent. AmB-LC (20 micrograms/ml) increased fMLP-stimulated chemiluminescence to two times that of PMNs plus MNLs without AmB-LC. AmB-des (2 micrograms/ml) (but not AmB-LC [2 micrograms/ml]) increased nitroblue tetrazolium reduction by PMNs in whole blood from 31 to 52% of positive cells. Neither form of AmB increased Mac-1 (the CD11b/CD18 integrin) expression of pure PMNs. AmB-des (0.5 to 2 micrograms/ml) (but not AmB-LC [< or = 40 micrograms/ml]) nearly doubled PMN Mac-1 expression in the presence of MNLs, and cell-free AmB-des (2 micrograms/ml)-stimulated, MNL-conditioned medium stimulated PMN Mac-1 to 125% of the control level. AmB-des (0.2 to 2 micrograms/ml) (but not AmB-LC [< or = 40 micrograms/ml]) decreased chemotaxis of pure PMNs to fMLP by as much as 35% and that of PMNs in the presence of MNLs by as much as 50%. Desoxycholate by itself had no effect on PMN function. These differences in activity between AmB-des and AmB-LC may explain the lessened toxicity observed with AmB-LC.     

Entry of gut lymph into the circulation primes rat neutrophil respiratory burst in hemorrhagic shock.

OBJECTIVE: Endothelial cell injury by polymorphonuclear neutrophil (neutrophil [PMN]) respiratory burst after trauma and hemorrhagic shock (T/HS) predisposes subjects to acute respiratory distress syndrome and multiple organ failure. T/HS mesenteric lymph injures endothelial cell and lymph duct ligation (LDL) before T/HS prevents pulmonary injury. We investigated the role of mesenteric lymph in PMN priming by T/HS. DESIGN: Prospective experiment in rats. SETTING: University hospital laboratory. SUBJECTS: Adult male rats. INTERVENTIONS: Mesenteric lymph was obtained from rats undergoing T/HS (30 mm Hg, 90 mins) or sham shock (T/SS). Plasma was harvested from uninstrumented control (UC), T/HS, T/SS, and T/HS+LDL rats. PMNs were isolated from UC, T/HS, and T/HS+LDL rats. MEASUREMENTS AND MAIN RESULTS: PMNs from UC rats were incubated in buffer, 1% T/HS lymph, and 1% T/SS lymph. PMNs from UC rats were incubated in UC, T/HS, T/SS, and T/HS+LDL plasma. PMN respiratory burst was initiated by using macrophage inflammatory protein (MIP)-2/platelet-aggregating factor (PAF) or phorbol myristate acetate. Cytosolic calcium ([Ca2+]i) responses to MIP-2/PAF were assayed in PMN from UC, T/HS, and T/HS+LDL rats. PMN preincubated in T/HS lymph showed significant elevations in MIP/PAF-elicited respiratory burst compared with T/HS lymph or buffer only (p <.05; analysis of variance/Tukey's test). T/HS lymph incubation also increased (p <.05) phorbol myristate acetate elicited respiratory burst compared with buffer or T/SS. Preincubation in T/HS plasma increased MIP-2/PAF-elicited respiratory burst (p <.05) compared with UC or T/SS plasma. LDL blocked T/HS priming of respiratory burst. Control PMN [Ca2+]i responses to MIP-2 and PAF were low. T/SS PMN were significantly more responsive, but the T/HS PMN showed still higher responses (p <.01). LDL reversed the priming of [Ca2+]i responses by T/HS (p <.01). CONCLUSIONS: PMNs are primed by T/HS lymph but not T/SS lymph and by T/HS plasma but not T/SS plasma. LDL before shock prevents T/HS plasma from priming PMN. The magnitude of respiratory burst found here paralleled the [Ca2+]i responses seen to receptor dependent initiating agonists. Mesenteric lymph is both necessary and sufficient to prime PMN after T/HS in the rat, and it primes PMN in part by enhancing [Ca2+]i responses to G-protein coupled chemoattractants. Mesenteric lymph mediates postshock PMN dysfunction.     

Oxygen radical-induced erythrocyte hemolysis by neutrophils. Critical role of iron and lactoferrin.

Human neutrophils (PMN), when stimulated with such chemotaxins as phorbol myristate acetate (PMA), destroy erythrocytes and other targets. Cytotoxicity depends on PMN-generated reactive oxygen metabolites, yet the exact toxic specie and its mode of production is a matter of some dispute. Using 51Cr-labeled erythrocytes as targets, we compared various reactive-O2 generating systems for their abilities to lyse erythrocytes as well as to oxidize hemoglobin to methemoglobin. PMA-activated PMNs or xanthine oxidase plus acetaldehyde were added to target erythrocytes in amounts that provided similar levels of superoxide. PMNs lysed 68.3 +/- 2.9% (SEM) of targets, whereas the xanthine oxidase system was virtually impotent (2.3 +/- 0.8%). In contrast, methemoglobin formation by xanthine oxidase plus acetaldehyde was significantly greater than that caused by stimulated PMNs (P less than 0.001). A similar dichotomy was noted with added reagent H2O2 or the H2O2-generating system, glucose plus glucose oxidase; neither of these caused 51Cr release, but induced 10-70% methemoglobin formation. Thus, although O2- and H2O2 can cross the erythrocyte membrane and rapidly oxidize hemoglobin, they do so evidently without damaging the cell membrane. That a granule constituent of PMNs is required to promote target cell lysis was suggested by the fact that agranular PMN cytoplasts (neutroplasts), although added to generate equal amounts of O2- as intact PMNs, were significantly less lytic to target erythrocytes (P less than 0.01). Iron was shown to be directly involved in lytic efficiency by supplementation studies with 2 microM iron citrate; such supplementation increased PMN cytotoxicity by approximately 30%, but had much less effect on erythrocyte lysis by neutroplasts (approximately 3% increase), and no effect on lysis in the enzymatic oxygen radical-generating systems. These results suggest a critical role for an iron-liganding moiety that is abundantly present in PMN, marginally so in neutroplasts, and not at all in purified enzymatic systems--a moiety that we presume catalyzes very toxic O2 specie generation in the vicinity of juxtaposed erythrocyte targets. The obvious candidate is lactoferrin (LF), and indeed, antilactoferrin IgG, but not nonspecific IgG, reduced PMN cytotoxicity by greater than 85%. Re-adding 10(-8) M pure LF to neutroplasts increased their ability to promote hemolysis by 48.4 +/- 0.9%--to a level near that of intact PMNs. We conclude that O-2 and H2O2 are not sufficient to mediate target cell lysis, but require iron bound to LF, which, in turn, probably generates and focuses toxic O2 radicals, such as OH, to target membrane sites.     

感染性休克患者血浆游离皮质醇浓度的变化分析

目的 探讨感染性休克患者在发病过程中血浆游离皮质醇浓度的变化情况.方法 对我院ICU收治的57例感染性休克患者的游离皮质醇浓度进行检测,观察其在感染性休克发病过程中的变化情况,并与31例非感染性危重病组及23例健康对照组进行比较.结果 与非感染的危重病组及健康对照组相比,感染性休克组中生存者的游离皮质醇浓度要明显高于其他两组,(2.21±0.45)nmol/L vs(1.12±0.48)nmol/L,(0.44±0.11)nmol/L(P<0.05);而感染性休克的死亡组浓度又高于存活组,(2.69±0.66)nmol/L.vs(2.21±0.45)nmol/L(P<0.05),对感染性休克存活者的分析发现,血浆游离皮质醇浓度随着病情的控制而降低,(2.214±0.45)nmol/L vs(1.52±0.38)nmol/L vs(0.57±0.31)nmol/L(P<0.05).结论 感染性休克患者的血浆游离皮质醇浓度和病情变化有关,可以作为判断预后的一项指标.     

Reduced neutrophil superoxide anion release after prolonged infusions of lidocaine

Lidocaine is used extensively in coronary care units, yet the effect of lidocaine infusions on neutrophil function has not been known. Lidocaine and other local anesthetics impair leukocyte antibacterial functions when added in vitro. We found that lidocaine added to human neutrophils in vitro markedly impaired the release of superoxide anion (O2-) and the granule enzymes lysozyme and myeloperoxidase after stimulation by phorbol myristate acetate or opsonized zymosan. We then measured production of O2- during stimulation of neutrophils from eight normal subjects, five coronary artery disease patients not receiving lidocaine and 13 coronary artery disease patients receiving lidocaine infusions for at least 12 hr. Release of O2- by cells from lidocaine-treated patients (14.2 +/- 3.8 nmol/2.5 X 10(6) neutrophils per 15 min) was significantly lower than from cells of the normal subjects (78.4 +/- 7.2 nmol; P less than .001) and the coronary patients not receiving lidocaine (70.6 +/- 4.0 nmol; P less than .001). Bactericidal assays at a high concentration (2 mg/ml) of lidocaine demonstrated slight reductions in 2 hr killing rates for Escherichia coli (70% with lidocaine vs. 95% control). Inhibition by lidocaine of the release of toxic oxygen metabolites from neutrophils could potentially reduce infarct size in patients with acute myocardial infarction; but as there is only a slightly reduced ability to kill bacteria, increased susceptibility to infections is unlikely although it cannot be excluded.